AMP-activated protein kinase (AMPK) is serine/threonine kinase that serves as an intracellular energy sensor and a key regulator of cell metabolism, growth and death, hence a promising drug target for obesity, diabetes, cancer and cardiovascular diseases. AMPK is a heterotrimeric complex composed of catalytic 1-subunit and regulatory 2- and 3-subunits with multiple isoforms for each subunit. Previous studies have shown that activation of AMPK in the heart during stress is an important cardioprotective mechanism. However, point mutations in the regulatory 32-subunit (encoded by prkag2 gene) of AMPK cause human cardiomyopathy characterized by cardiac hypertrophy, arrhythmias and glycogen storage. This raises concerns of future drug development targeting AMPK cascade in the heart. It is thus critical to understand the disease mechanisms responsible for the prkag2 cardiomyopathy. Using transgenic mice overexpressing mutant prkag2 (N488I) in the heart (TG32N488I) that faithfully recapitulated prkag2 cardiomyopathy, we in the previous funding period demonstrated that the mutation caused aberrant activation of AMPK in the absence of energetic deficit, and the cardiomyopathy phenotype could be rescued by inhibition of AMPK activity. We have further demonstrated that the simultaneous increases in glucose uptake and fatty acid oxidation in the absence of energy stress resulted in metabolic re-routing of exogenous glucose preferentially into glycogen pool in the TG32N488I hearts. To determine whether the cardiomyopathy phenotype is entirely attributable to glycogen storage, we sought to rescue the glycogen storage without changing the mutant gene expression by specifically targeting the activity of muscle glycogen synthase. Our preliminary data showed that this strategy successfully normalized cardiac glycogen content but not the cardiac hypertrophy phenotype in TG32N488I mice. Thus, we hypothesize that 32- AMPK mutation causes cardiac hypertrophy independent of glycogen storage. Moreover, since the prkag2 mutation causes predominantly cardiac phenotype, it raises the question of a unique role of 32-AMPK in the heart. This led us to generate mouse models deficient of 32-AMPK in order to determine the isoform-specific function of 3-AMPK. Using these models we propose 1) to test the hypothesis that the prkag2 mutation causes cardiac hypertrophy independent of glycogen storage and, to determine the mechanisms by which aberrant activation of 32-AMPK under nutrient saturated conditions stimulate cardiac hypertrophy; 2) to define the composition and the function of the 32-AMPK signaling cascade in the heart during cardiac development and diseases.